Reference is made to U.S. application Ser. No. 09/837,946, filed Apr. 19, 2001, entitled xe2x80x9cAutomated Computer Controlled Reporter Device for Conducting Immunoassay and Molecular Biology Procedures.xe2x80x9d
This invention relates to detection of bioagents (and/or nucleic acids), and, more particularly, to a method for identifying the presence of bioagents and/or nucleic acids of specific interest in a specimen in the shortest possible time, with a minimum of necessary equipment and using known tests.
Health authorities are ever alert to the outbreak of a contagious disease that, if not checked early, could grow to epidemic proportion amongst the general population. To that purpose, the health authorities are charged with monitoring the environment for the incidence of unusual illnesses, symptomatic of a disease, obtain and analyze samples to identify the biological agent causing the disease, and, once identified, publish protective measures that must be taken to halt the spread of the disease. Typically, biological agents, such as virus and bacteria, are part of nature, and outbreaks of disease occur naturally. However, biological terrorism, the deliberate release of a harmful virus or bacteria or other biological agent (hereafter, xe2x80x9cbioagentxe2x80x9d) amongst a general population, and bio-warfare, the deliberate release of a bioagent against military troops in battle, are also of concern.
One known test procedure or process for detection of a specific bioagent that is applicable to a variety of fields, such as biotechnology, environmental protection and public health, is the enzyme linked immunoassay (hereafter referred to as xe2x80x9cELISAxe2x80x9d). The ELISA process constitutes an identification process that uses molecular interactions to uniquely identify target substances. A basic definition of ELISA is a quantitative in vitro test for an antibody or antigen (e.g., a bioagent) in which the test material is adsorbed on a surface and exposed to a complex of an enzyme linked to an antibody specific for the substance being tested for with a positive result indicated by a treatment yielding a color in proportion to the amount of antigen or antibody in the test material. The basic ELISA procedure is described more specifically, for one, in a book entitled Methods in Molecular Biology Vol 42, John R. Crowther, Humana Press, 1995.
The xe2x80x9cantibody specific for the substance being tested forxe2x80x9d in the foregoing definition constitutes a recognition molecule, a molecule that is capable of binding to either reactant or product molecules in a structure-restricted manner. That is, the recognition molecule binds to a specific three-dimensional structure of a molecule or to a two-dimensional surface that is electrically charged and/or hydrophobic in a specific surface pattern. It may also be recognized that ELISA-like approaches using other recognition molecules can also be used, such as aptamers, DNA, RNA and molecular imprint polymers.
More recently, the foregoing definition for ELISA has been expanded beyond the colormetric approach, wherein color and color intensity is used as a reporter or indicia of the antigen or antibody, to include a voltametric or amperiometric approach to detection, wherein a rate of change of voltage or current conductivity in proportion to the amount of antigen or antibody contained in the test material. Patent Cooperation Treaty application PCT/US98/16714, filed Aug. 12, 1998 (International Publication No. WO 99/07870), entitled xe2x80x9cElectrochemical Reporter System for Detecting Analytical Immunoassay and Molecular Biology Proceduresxe2x80x9d (hereafter the xe2x80x9c16714 PCT applicationxe2x80x9d), claiming priority of U.S. patent application Ser. Nos. 09/105,538 and 09/105,539xe2x80x9d), to which the reader may refer, describes both a colormetric and an electrochemical reporter system for detecting and quantifying enzymes and other bioagents in analytical and clinical applications. The electrochemical reporter system of the 16714 PCT application employs a sensor for detecting voltametric and/or amperiometric signals that are produced in proportion to the concentration of organic (or inorganic) reporter molecules by redox (e.g. reduction-oxidation) recycling at the sensor.
In brief, in the ELISA test, the suspect bioagent is initially placed in a water-based buffer, such as a phosphate buffered saline solution, to form a sample solution. That sample solution is mixed with a quantity of particles, beads, the surface of which is coated with an antibody to the suspect bioagent, a recognition molecule (also sometimes referred to as a receptor molecule). The particular antibodies used to coat the beads are known to bind to the bioagent of interest or of concern and is a primary antibody or xe2x80x9c1o Abxe2x80x9d. That is, the antibody coating exhibits a chemical xe2x80x9cstickinessxe2x80x9d that is selective to specific bioagents.
Any bioagent present in the sample solution binds with a non-covalent bond to a respective antibody and thereby becomes attached to a respective one of the beads in the mixture-solution. If the sample solution does not contain a bioagent or if the bioagent that is present in the solution is not one that binds to the selected antibody, then nothing binds to the foregoing antibody. Further processing of the ELISA process then shows nothing.
Assuming the suspect bioagent is present in the sample, the bioagent bonds to the antibody that is coated on the beads. The solution then contains a quantity of bioagent molecules bound respectively to a like quantity of coated beads. The mixture is optionally washed, as example, in a phosphate-buffered saline, and a second antibody, more specifically, an antibody and enzyme linked combination, is then added to the mixture. The second antibody is also one that is known to bind to the suspect bioagent, another recognition molecule. The second antibody may be either be one that is monoclonal, e.g. one that binds to only one specific molecule, or polyclonal, e.g. a mixture of different antibodies each of which shares the characteristic of bonding to the target bioagent. The enzyme, is covalently bound to the second antibody and forms a complex that is referred to as a secondary antibody-enzyme conjugate or xe2x80x9c2oAb-enzxe2x80x9d. As known by those skilled in the art, an enzyme is a xe2x80x9cmolecule scissorsxe2x80x9d, a protein that catalyzes a biological reaction, a reaction that does not occur appreciably in the absence of the enzyme. The enzyme is selected to allow the subsequent production of an electrochemically active reporter.
The 2oAb-enz binds to the exposed surface of the immobilized bioagent to form an xe2x80x9cantibody sandwichxe2x80x9d with the bioagent forming the middle layer of that sandwich. The antibody sandwich coated beads are washed again to wash away any excess 2oAb-enz in the solution that remains unbound.
The beads and the attached antibody sandwich, the 1oAb/bioagent/2oAb-enz complex, in the solution are placed over the exposed surface of the redox recycling sensor. The substrate of the foregoing enzyme is added to the solution and the substrate is cleaved by the enzyme to produce an electrochemically active reporter. The substrate of the enzyme, referred to as PAP-GP, is any substance that reacts with an enzyme to modify the substrate. The effect of the enzyme is to separate, cut, the PAP, a para-amino phenol, the electrochemically active reporter, from the GP, an electrochemically inactive substance.
The foregoing chemical reaction is concentrated at the surface of the sensor. The rate of production of the foregoing reporter (PAP) is proportional to the initial concentration of bioagent. The reporter reacts at the surface of the sensor, producing an electrical current through the sensor that varies with time and is proportional to the concentration of the bioagent, referred to as redox recycling. The occurrence of the electric current constitutes a positive indication of the presence of the suspect bioagent in the sample. Analysis of the electric currents produced over an interval of time and comparison of the values of that electric current with existing laboratory standards of known bioagents allows quantification of the concentration of bioagent present in the initial sample.
As recognized, the greater the number of laboratories equipped to check for specific bioagents and the more widely those laboratories are dispersed over a geographic region, the more swiftly a bioagent occurring in the environment can be recognized and handled by the health authorities. Unfortunately, the number of such facilities is relatively small. Recognizing that greater dispersal of ELISA test capability among the general population is a desirable goal in bioagent detection, it is also recognized that a lack of trained personnel is problematic, and that wide dispersal of ELISA test capability may not as a practical matter be possible unless the ELISA test can be carried out by persons of lesser skill.
As example, the electrochemical ELISA procedure and apparatus of the cited 16714 PCT application and the predecessor ELISA procedures appear well suited to practice in a microbiology laboratory by highly skilled personnel who are alert to the details of the test process. Other facilities or environments in which such an analysis is desirable, however, may not enjoy either the availability of highly skilled technicians or an adequately equipped laboratory. In such environments, the availability of a foolproof, user-friendly test apparatus that is able to analyze a sample and report a meaningful result with minimal human intervention is certainly desirable.
Recognizing that need, the present inventors, together with other co-inventors, invented an automated test procedure, which is described in U.S. patent application Ser. No. 09/837,946, filed Apr. 19, 2001, entitled Automated Computer Controlled Reporter Device for Conducting Immunoassay and Molecular Biology Procedures (hereafter the xe2x80x9c946 applicationxe2x80x9d), assigned to the assignee of the present application, the content of which is incorporated herein by reference. The apparatus of the ""946 application provides a user friendly stand-alone portable unit that is able to automatically perform an ELISA test which may be operated by persons who are not biologists and who require minimal training. The automated system contains a number of solutions and pumps that are controlled by a programmed computer.
The foregoing system also employs beads of magnetic material and a magnetic positioning device to manipulate and position the coated magnetic beads under control of the computer, such as during the washing steps of the ELISA process, and in positioning the beads at the sensor during redox recycling. The automated test device of the ""946 application provides a solution to dispersal of testing units that takes into account the lesser skills of prospective operators. However, like the prior manual procedure, the automated procedure is unable to determine which suspected virus should be sought or the priority to employ in the search to identify the suspect bioagent.
Each of the foregoing ELISA test procedures, whether manual or automatic, and/or colormetric or amperiometric, searches for a single suspect bioagent, and, if detected, determines the concentration of that bioagent. The identification is essentially a xe2x80x9cgoxe2x80x9d or xe2x80x9cno-goxe2x80x9d procedure. In one approach to identification, the test procedure is repeated using different receptor molecules until the bioagent is identified. If the result of the one test is a xe2x80x9cno goxe2x80x9d, then a second bioagent is made the suspect and the test is repeated for that second bioagent. The foregoing test procedure may be continued almost indefinitely until the particular bioagent is detected, one exhausts the supply of known receptor molecules or one exhausts the supply of known bioagents.
Even though the ELISA test is automated, as when employing the test apparatus of the cited ""946 application, identification of a bioagent could take a great deal of time to accomplish if testing is accomplished in serial order, particularly if the bioagent turns out to be the least likely one in an extended list of suspect bioagents. One solution for reducing the time to identification is to utilize a bank of test apparatus, one test apparatus for each bioagent in the group of possibilities, and carry out the separate ELISA test procedures concurrently. Such an approach requires a great deal of equipment, particularly if one tests for a great number of different bioagents, and appears to be impractical. As example, if one is concerned about fifteen different bioagents as possibilities, it is possible to concurrently test using a bank of fifteen testers or other test apparatus, each catered to a respective bioagent. But so much test apparatus is expensive, requires a great deal of space in total, and takes much effort to maintain. Hence the approach is thought to be impractical.
Accordingly, an object of the present invention is to reduce the number of individual tests that must be performed to identify the presence of one of multiple suspect bioagents in a sample, and to reduce the time required to make such identification.
A further object of the invention is to provide the means to search for bioagents with increased speed and efficiency.
And, a still further object of the invention is define a testing protocol for application of the ELISA process.
In accordance with the foregoing objects, the existence of any one of up to 2Nxe2x88x921 bioagents in a sample is determined and identified by dividing the sample into N parts and performing N separate identification processes (e.g., ELISA), one process for each of the N parts, where N is an integer greater than 1. Each of those N identification processes is assigned a respective group or combination of bioagents to identify that is unique to the respective identification process, with the bioagents of the group or combination being selected from the 2Nxe2x88x921 bioagents and with the sum of those bioagents constituting the group or combination being less than 2Nxe2x88x921 bioagents.
Each such identification process is capable of identifying any one of a number of bioagents in the group or combination of bioagents assigned for detection to the respective identification process. At least some of the bioagents in the group or combination of bioagents assigned to one identification process are also shared by the group or combination of bioagents that are assigned for identification to at least one other one of the identification processes and each group or combination is assigned a bioagent that is unique to the respective identification process. Each of the N separate identification processes accordingly possess the capability of uniquely identifying a respective single one of the bioagents from the 2Nxe2x88x921 bioagents combination that none of the other identification processes is capable of identifying. By use of combinational logic a particular bioagent may be identified.
In one embodiment in which N equals the number two, the number of bioagents that can be detected using two ELISA processes is three. Thus the sample containing one of the bioagents (or none) is parsed in two and separately tested for the bioagents. The one ELISA process being capable of identifying only bioagents A and B and the other ELISA process being capable of identifying only bioagents B and C, whereby the identification of bioagent B, common to both combinations, is shared. Further, the one process is uniquely capable of identifying bioagent A, the other process is uniquely capable of identifying bioagent C. Thus, if both identification processes identify a bioagent, combination logic dictates that the bioagent is B. Otherwise only one of the two identification processes will identify bioagent A or C if present. As an advantage, the new process permits rapid identification of one of several bioagents with fewer than several tests and/or equipment for several tests.
The foregoing and additional objects and advantages of the invention, together with the structure characteristic thereof, which were only briefly summarized in the foregoing passages, will become more apparent to those skilled in the art upon reading the detailed description of a preferred embodiment of the invention, which follows in this specification, taken together with the illustrations thereof presented in the accompanying drawings.